In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of the rat mucosal mast cell tumor line RBL after transfection with genes for cytotoxic lymphocyte granule components. In particular we have sought to test whether the granule serine proteases known as granzymes play a role in target DNA degradation accompanying cytotoxicity. We have constructed double and single RBL transfectants expressing cytolysin (cy) and the granule serine protease granzyme A (gza). RBL-cy transfectants show potent killing on red blood cell targets, and moderate cytotoxicity on tumor targets. However, DNA degradation in the latter is negligible. While RBL-gza transfectants express gza at levels comparable to cloned CTL and secrete it in response to IgER crosslinking, they have no cytotoxic activity detectable. RBL-cy-gza transfectants showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on both RBC and tumor targets. Target DNA breakdown by these double transfectants was clearly positive, with dose response curves parallel to lysis but somewhat less efficient. Agarose gels of labelled target DNA showed the ladder pattern characteristic of internucleosomal cleavage. These results clearly show that granzyme A can play a role in target DNA breakdown. As a direct test of the ability of proteases to induce cytotoxicity when introduced into the cytoplasm of a target cell, we have "injected" various proteases into tumor cells using osmotic lysis of pinosomes. The endoproteases trypsin, chymotrypsin, proteinase K, and papain were all found to lyse lymphoma cells in a dose dependent manner, as measured by 5ICr release; this measure of death was preceded by DNA release. Microscopically, these cells were shown to undergo a marked membrane blebbing and shrinking, characteristic of "apoptosis".